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    2019-09-23

    r> Because all responses occurred in the HR-deficient subset, we assessed whether HRD score correlated with percent change of tumor area from baseline in this subset of patients. No correlation was ob-served (Fig. 4A, Spearman r = 0.050; p = 0.87). Thus, while a positive HRD test enriched for responders, the actual value of the score provided no additional information regarding the extent of response.
    3.4. Relationship between PARP1 Necrosulfonamide and response
    In view of preclinical studies described above suggesting that differ-ences in repair pathways can affect PARPi sensitivity, we stained the cancer specimens from this trial for a series of DNA repair proteins. When the relationship between response and staining intensity was ex-amined across the entire study population, there was a statistically sig-nificant association between HRD status and response, as anticipated, but no significant correlation between protein H-scores and response (Table 1). Because previous reports have demonstrated the role of NHEJ in vitro and in vivo specifically in the setting of HR deficiencies [11-16], we next performed a preplanned analysis examining the rela-tionship between repair protein levels and response in HR-deficient and HR-proficient carcinomas (as defined by the Myriad HRD assay) separately.
    When we examined the relationship between response and PARP1, a protein whose loss is associated with poor PARPi response in cell lines [9,10,35], no association was observed between PARP1 expression and clinical response (p = 0.203, Fig. S5A). Accordingly, when the relation-ship between PARP1 H-score and percent change in tumor cross sec-tional area from baseline was examined specifically in the HR-
    Fig. 1. 53BP1 loss and PARP inhibitor sensitivity in HR-deficient ovarian cancer cells. A, Expression of 53BP1 of was assessed by immunoblotting in whole cell lysates of COV362 transduced with empty vector (EV) or sgRNA targeting 53BP1 (53BP1−/−) as well as OV90 and PE01 cells. B, Homologous recombination repair was assayed using the DR-GFP plasmid as described in the Methods and illustrated in Fig. S1. OV90 (HR proficient—Ref. [24]) and PE01 cells (HR deficient—Refs. [11, 23]) served as controls for this assay. Error bars, summary of 4 independent assays. PARPi sensitivity of the positive and negative controls is shown in Fig. S1C. C, COV362 empty vector (EV) and COV362 53BP1−/− cells were continuously exposed to increasing concentrations of PARP inhibitor veliparib in a clonogenic assay. Error bars, ± SEM from triplicate plates in a single assay. Across 5 independent assays, the IC50 for veliparib was 2.9 ± 0.4-fold (mean ± SEM) higher in the 53BP1−/− cells.
    Fig. 2. Validation of 53BP1 antibody staining. OVCAR8 cells transfected with nontargeting siRNA (control siRNA) or siRNA targeting 53BP1 (53BP1 siRNA) were subjected to immunoblotting (A) or formalin fixed, embedded in paraffin, and stained to confirm specificity of the anti-53BP1 antibody (B, C). D, cores from the same ovarian cancers included on two different TMAs were independently stained with anti-53BP1 and scored. E, 53BP1 staining of four ovarian cancers to illustrate range of expression in the present study.
    3.5. Relationship between 53BP1 expression and response
    In contrast, in HR-deficient carcinomas, there was a strong negative correlation between 53BP1 H-score and percent change of tumor area from baseline (Fig. 4B, left panel; r = −0.69, p = 0.004), consistent with pre-clinical studies showing that 53BP1 loss in BRCA1-mutant mu-rine cells is associated with PARPi resistance [13–15]. Additionally, there was a trend toward increased shrinkage of tumor from baseline and higher KU80 H-score (Fig. 4C, left panel; r = −0.46, p = 0.08) or higher DNA-PKCS H-score (Fig. 4D, left panel; r = −0.38; p = 0.17). In contrast, no significant correlations were observed between 53BP1, KU80 or DNA-PKCS expression and change in tumor size among the HR-proficient tumors (Fig. 4B–D, right panels). Moreover, no significant cor-relations were observed in either HR-deficient or HR-proficient carcino-mas between KU70, XRCC4, or RAD51 H-scores and percent change of tumor area (Fig. S6).